PROJECT SUMMARY Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes a range of diseases and is one of the major infectious agents responsible for ventilator-associated pneumonia. In order for A. baumannii to cause disease, it must acquire nutrient zinc from its environment and compete with the host for this essential metal. Keeping with this, A. baumannii upregulates a variety of zinc acquisition genes in response to nutrient limitation. A highly induced gene during zinc limitation encodes a putative D-alanine-D-alanine carboxypeptidase which we have named zrlA, which is an enzyme class involved in peptidoglycan remodeling. While these carboxypeptidases are broadly classified as being involved in cell wall maintenance, their importance in bacterial pathogenesis and their contribution to nutrient acquisition is not well-defined. Genetic inactivation of zrlA results in a significant growth defect during zinc limitation and reduces fitness in a murine model of pneumonia. Furthermore, this strain is extremely sensitive to antibiotic exposure, suggesting that ZrlA contributes to both nutrient zinc homeostasis and maintaining the barrier function of the cellular envelope. Therefore, we predict that ZrlA is specifically induced during zinc starvation to modify peptidoglycan in an effort to promote zinc acquisition and provide resistance to extracellular stress. This prediction will be tested through two specific aims. In Specific Aim 1, the contribution of ZrlA to overcoming zinc starvation will be determined by assessing alterations in zinc acquisition systems and uptake. Experiments proposed in Specific Aim 2 will more specifically dissect the impact of ZrlA on peptidoglycan architecture and cellular integrity. This aim will combine biochemical techniques with advanced microscopy to determine structural components of the bacterial cell envelope and the niche in which ZrlA contributes to this shape. Taken together, these findings will have broad implications for understanding the mechanism bacterial pathogens employ to acquire zinc despite nutrient limitation within the vertebrate host, and provide exciting targets for antimicrobial therapy development involving bacterial metal acquisition.